Protocol for risk stratification of ischemic events and optimized individualized treatment

ABSTRACT

A hemostasis analyzer, such as the Thrombelastograph® (TEG®) hemostasis analyzer is utilized to measure continuously in real time, the hemostasis process from the initial fibrin formation, through platelet-fibrin interaction and lysis to generate blood hemostasis parameters. The measured blood hemostasis parameters permit preparation of an individualized assessment of ischemic event risk and individualized treatment of a subject.

CROSS-REFERENCE TO RELATED APPLCATIONS

This application is a continuation of U.S. patent application Ser. No.12/347,466, filed Dec. 31, 2008 entitled Protocol for RiskStratification of Ischemic Events and Optimized IndividualizedTreatment, now U.S. Pat. No. 7,754,489; which is a continuation of U.S.patent application Ser. No. 11/753,071 filed May 24, 2007 entitledProtocol for Monitoring Direct Thrombin Inhibition, which is acontinuation-in-part of U.S. patent application Ser. No. 11/608,174filed Dec. 7, 2006 entitled Method of Evaluating Patient Haemostasis,now U.S. Pat. No. 7,732,213; which is a continuation-in-part of U.S.patent application Ser. No. 10/384,345 filed Mar. 7, 2003 entitledProtocol for Monitoring Platelet Inhibition, now U.S. Pat. No.7,179,652; which is a continuation-in-part of U.S. patent applicationSer. No. 09/591,371 filed Jun. 9, 2000 entitled Method and Apparatus forMonitoring Anti-Platelet Agents, now U.S. Pat. No. 6,613,573, which is acontinuation-in-part of U.S. patent application Ser. No. 09/255,099,filed Feb. 22, 1999, entitled Method and Apparatus for MeasuringHemostasis, now U.S. Pat. No. 6,225,126, the disclosures of which arehereby expressly incorporated herein by reference.

TECHNICAL FIELD

This patent relates to protocols for monitoring patient hemostasis andin particular protocols for an individualized assessment of ischemicevent risk and optimized individualized treatment of a subject.

BACKGROUND

Blood is the circulating tissue of an organism that carries oxygen andnutritive materials to the tissues and removes carbon dioxide andvarious metabolic products for excretion. Whole blood consists of a paleyellow or gray yellow fluid, plasma, in which are suspended red bloodcells, white blood cells, and platelets.

An accurate measurement of hemostasis, i.e., the ability of a patient'sblood to coagulate and dissolve, in a timely and effective fashion iscrucial to certain surgical and medical procedures. Accelerated (rapid)and accurate detection of abnormal hemostasis is also of particularimportance in respect of appropriate treatment to be given to patientsbeing prepared for, undergoing or recovering from surgical procedures orsuffering from hemostasis disorders and to whom it may be necessary toadminister anticoagulants including direct or indirect thrombininhibitors, antifibrinolytic agents, thrombolytic agents, anti-plateletagents, or blood components in a quantity which must clearly bedetermined after taking into account the circumstances of the surgeryand/or the abnormal components, cells or “factors” of the patient'sblood which may be contributing to the hemostasis disorder.

Hemostasis analyzer instruments have been known since Professor HelmutHartert developed such a device in Germany in the 1940's. One type ofhemostasis analyzer is described in U.S. Pat. No. 5,223,227, thedisclosure of which is hereby expressly incorporated herein byreference. This instrument, the TEG® hemostasis analyzer, monitors theelastic properties of blood as it is induced to clot under a low shearenvironment resembling sluggish venous blood flow. The patterns ofchanges in shear elasticity of the developing clot enable thedetermination of the kinetics of clot formation, as well as the strengthand stability of the formed clot; in short, the mechanical properties ofthe developing clot. As described above, the initial fibrin formation,kinetics, strength and stability of the clot provides information aboutthe ability of the clot to perform “mechanical work,” i.e., resistingthe deforming shear stress of the circulating blood; in essence, theclot is the elementary machine of hemostasis, and the TEG® analyzermeasures the ability of the clot to perform mechanical work throughoutits structural development. The TEG® system measures continuously allphases of patient hemostasis as a net product of whole blood componentsin a non-isolated, or static fashion from the time of test initiationuntil initial fibrin formation, through clot rate strengthening andultimately clot strength through fibrin platelet bonding via plateletGPIIb/IIIa receptors and clot lysis.

Normal hemostasis process results in a three-dimensional network ofpolymerized fibrin fibers which together with platelet glycoproteinIIb/IIIa (GPIIb/IIIa) receptor bonding forms a final clot (FIG. 1 a). Aunique property of this network structure is that it behaves as a rigidelastic solid, capable of resisting deforming shear stress of thecirculating blood. The strength of the final clot to resist deformingshear stress is determined by the structure and density of the fibrinfiber network and by the forces exerted by the participating platelets.

The clot that develops and adheres to the damaged vascular system as aresult of activated hemostasis and resists the deforming shear stress ofthe circulating blood is, in essence a mechanical device, formed toprovide a “temporary stopper,” that resists the shear force ofcirculating blood during vascular recovery. The initial fibrinformation, kinetics, strength, and stability of the clot, that is itsphysical property to resist the deforming shear force of the circulatingblood, determine its capacity to do the work of hemostasis, which is tostop hemorrhage without permitting inappropriate thrombosis. This isexactly what the Thrombelastograph® (TEG®) system was designed tomeasure, which is the time it takes for initial clot formation, the timeit takes for the clot to reach its maximum strength, the actual maximumstrength, and the clot's stability.

Thrombin is an enzyme that cleaves soluble fibrinogen into fibrinstrands. It is also the most potent platelet activator and strongly anddirectly increases the expression and activation of platelet GPIIb/IIIareceptors. Platelets and fibrin cooperate to increase the mechanicalstrength of the clot in at least two ways. First, platelets provide nodebranching points to which fibrin strands attach, significantly enhancingclot structural rigidity. Secondly, the platelets exert a “tugging”force on the fibrin fibers, by the contractibility of plateletactomyosin, a muscle protein that is a part of a cytoskeleton-mediatedcontractibility apparatus. The force of this contractibility furtherenhances the strength of the fibrin/platelet structure and hence theresulting clot. Thus, thrombin's role in the hemostasis process, and inparticular in mediating thromboembolic complications, is clear.

Despite a rather narrow therapeutic dosing range and a lack of a readyantidote, bivalirudin, a direct thrombin inhibitor, is being more widelyused in percutaneous coronary interventional (PCI) procedures in placeof heparin (an indirect thrombin inhibitor) since it has a morepredictable anticoagulant effect. However, current methodologies such asthe standard ACT tests based on kaolin do not accurately reflectanticoagulation by bivalirudin at higher doses raising the possibilityof over-dosing patients. Ecarin based tests have been suggested as beingbetter than ACT or other standard coagulation tests since ecarindirectly activates prothrombin to a miezo-thrombin form, which has lessfeedback procoagulant activity than thrombin.

Along with thrombin, platelets play a critical role in mediatingischemic complications in prothrombotic (thrombophilic) patients. Theuse of GPIIb/IIIa inhibitor agents in thrombophilic patients or as anadjunct to PCI is rapidly becoming the standard of care. Inhibition ofthe GPIIb/IIIa receptor is an extremely potent form of anti-platelettherapy that can result in reduction of risk of death and myocardialinfarction, but can also result in a dramatic risk of hemorrhage. Thereason for the potential of bleeding or non-attainment of adequatetherapeutic level of platelet inhibition is the weight-adjusted plateletblocker treatment algorithm that is used in spite of the fact that thereis considerable person-to-person variability. This is an issue in partdue to differences in platelet count and variability in the number ofGPIIb/IIIa receptors per platelet and their ligand binding functions.

Since the clinical introduction of the murine/human chimeric antibodyfragment c7E3 Fab (abciximab, ReoPro®), several synthetic forms ofGPIIb/IIIa antagonists have also been approved such as Aggrastat®(tirofiban) and Integrilin® (eptifibatide), resulting in widespread andincreasing use of GPIIb/IIIa inhibitor therapy in interventionalcardiology procedures.

Before the introduction of the method and apparatus described in theafore-mentioned U.S. Pat. No. 6,613,573, there was no rapid, reliable,quantitative, point-of-care test for monitoring therapeutic plateletblockade. Although the turbidimetric aggregometer test has been used tomeasure the degree of platelet GPIIb/IIIa receptor blockade in smallclinical studies and dose-finding studies, its routine clinical use fordosing GPIIb/IIIa receptor antagonists in individual patients has notbeen feasible. Measurement by aggregometer is time-consuming (more thanone hour), expensive to run, requires specialized personnel for itsperformance, and is not readily available around the clock; therefore itcannot be employed at the point-of-care for routine patient monitoringand dose individualization. To be clinically useful, an assay ofplatelet inhibition must provide rapid and reliable informationregarding receptor blockade at the bedside, thereby permitting dosemodification to achieve the desired anti-platelet effect.

The turbidimetric aggregometer test is based on the photometricprinciple, which monitors the change in the specimen's optical density.Initially, a minimal amount of light passes through the specimen, asfunctional platelets are activated by the turbidimetric test; plateletaggregation occurs via platelet GPIIb/IIIa receptor and fibrin(ogen)bonding as illustrated in FIG. 1 a, and thus light transmissionincreases. When platelets are inhibited through GPIIb/IIIa receptorblockade, light transmission decreases proportionally.

Another commercially available system measures fibrinogen-plateletbonding using beads coated with a fixed amount of an outside source of“normal” fibrinogen. Therefore, this system uses a non-patient source of“normal” fibrinogen and cannot detect a patient in a prothrombotic state(hypercoagulable) due to a higher patient level of fibrinogen, or detecta hemorrhagic state (hypocoagulability) due to a low patient level offibrinogen. Additionally, this system shows only bonding withoutdetection of the breakdown of that bonding. Therefore, in the presenceof thrombolysis, the assessment of platelet GPIIb/IIIa receptor blockadeby the system may not be accurate.

Fibrinogen-platelet GPIIb/IIIa bonding (FIG. 1 a) is the initial phaseof platelet aggregation, or a primary hemostasis platelet plug, which isreversible; this goes on to form the final fibrin-platelet bonding (FIG.1 b). Thus it is not sufficient to measure only the initial stage offibrinogen-platelet bonding, which may not accurately reflect finalfibrin-platelet bonding via the GPIIb/IIIa receptor. While theturbidimetric and other photometric systems do detect initiation ofplatelet aggregation via fibrinogen-platelet GPIIb/IIIa receptor bonding(FIG. 1 a), it may not accurately reflect final fibrin-platelet bondingvia the GPIIb/IIIa receptor, which is non-reversible (FIG. 1 b).

Significant among the limitations of systems that use beads coated with“normal” fibrinogen is that this “normal” fibrinogen may not reflecteither the quantity or the functionality of a specific patient's ownfibrinogen. Therefore, fibrinogen-platelet GPIIb/IIIa receptor blockadeas measured by such systems is but a rough estimate of the patient'sindividual fibrinogen-platelet GPIIb/IIIa blockade of the initial phaseof platelet aggregation.

This is a significant limitation in certain high risk patient subgroups,which may need treatment with a platelet inhibition agent, may have ahigher or lower level of fibrinogen and thus would need an accurateassessment of platelet GPIIb/IIIa receptor blockade to reduce bleedingcomplications due to under assessment of platelet GPIIb/IIIa receptorblockade, or ischemic events due to over assessment of plateletGPIIb/IIIa receptor blockade. In addition, fibrinogen level andfunctionality may change during the trauma of interventional procedures.At this time it is imperative to make an accurate assessment of plateletGPIIb/IIIa receptor blockade in real time, during and following theprocedure.

Thus, there is a need for a method and apparatus for evaluatingcontributors to patient hemostasis both in the presence and absence oftherapies affecting hemostasis such as platelet inhibiting agents in thecase of platelet hypercoagulability, thrombin inhibiting agents in thecase of enzymatic hypercoagulability, and the like.

BRIEF DESCRIPTOIN OF THE DRAWINGS

FIG. 1 a is graphic illustration representing the initial phase ofplatelet aggregation.

FIG. 1 b is a graphic illustration representing fibrin-platelet bondingin final clot formation.

FIG. 2 is a schematic diagram of a hemostasis analyzer in accordancewith a preferred embodiment of the invention.

FIG. 3 is a plot illustrating a hemostasis profile generated by thehemostasis analyzer shown in FIG. 2.

FIGS. 4-10 are schematic illustrations depicting platelet activationresponsive to various agonist agents.

FIG. 11 illustrates several hemostasis profiles relating to the plateletactivation described in connection with FIGS. 4-10.

FIG. 12 is a schematic diagram of a hemostasis analyzer in accordancewith an alternate embodiment of the invention.

FIG. 13 is a chart illustrating initial clot formation times from asampling of patients before and after administration of ananticoagulation therapy.

FIGS. 14 a and 14 b are hemostasis profiles of a subject before andafter administration of an anticoagulation therapy.

DETAILED DESCRIPTION

In accordance with the preferred embodiments of the invention, ahemostasis analyzer, such as the Thrombelastograph® (TEG®) hemostasisanalyzer available from Haemoscope Corp., Niles, Ill., is utilized tomeasure continuously in real time, the hemostasis process from theinitial fibrin formation, through platelet-fibrin GPIIb/IIIa bonding andlysis. While several specific hemostasis therapies are discussed hereinin connection with the preferred embodiments of the invention, such asanti-platelet agent, direct or indirect thrombin inhibition, etc., itwill be appreciated the invention has application in connection withvirtually any hemostasis therapy. Moreover, it will be furtherappreciated that the invention has application for measuring theefficacy of coagulation enhancing agents.

In accordance with the preferred embodiments of the invention,utilization of the hemostasis analyzer in accordance with the inventiveprotocol permits: confirmation of the attainment of therapeutic level ofa hemostasis therapy; individualized dosing assessments; illustration ofthe rate of enhancement or diminishment of the therapy withadministration and evaluation of the interaction effect of a combinationof therapies on patient hemostasis.

The present invention may use a hemostasis analyzer 10, such as theThrombelastograph® (TEG®) hemostasis analyzer referenced above, tomeasure the clot's physical properties. An exemplary hemostasis analyzer10 is described in detail in the aforementioned U.S. Pat. No. 6,225,126,and a complete discussion is not repeated here. Alternative devices formeasuring hemostasis may be used such as those using contactless clotstrength measurement as shown in U.S. patent application Ser. No.11/383,567, now abandoned, or sample resonant excitation as shown inU.S. patent application Ser. No. 11/425,818, now U.S. Pat. No.7,879,615, the disclosures of which are hereby expressly incorporatedherein by reference. Any suitable whole blood analysis technique may beemployed that analyzes the time to clot formation, rate of clotformation, clot strength and lysis.

With reference to FIG. 2, to assist in the understanding of theinvention, however, a brief description of the hemostasis analyzer 10 isprovided. The hemostasis analyzer uses a special stationary cylindricalcup 12 that holds a blood sample 13. The cup 12 is coupled to a drivemechanism that causes the cup to oscillate through an angle θ,preferably about 4° 45′. Each rotation cycle lasts 10 seconds. A pin 14is suspended in the blood sample 13 by a torsion wire 15, and the pin 14is monitored for motion. The torque of the rotating cup 12 istransmitted to the immersed pin 14 only after fibrin-platelet bondinghas linked the cup 12 and pin 14 together. The strength of thesefibrin-platelet bonds affects the magnitude of the pin motion, such thatstrong clots move the pin 14 directly in phase with the cup motion.Thus, the magnitude of the output is directly related to the strength ofthe formed clot. As the clot retracts or lyses, these bonds are brokenand the transfer of cup motion is diminished.

The rotational movement of the pin 14 is converted by a transducer 16 toan electrical signal, which can be monitored by a computer (not shown inFIG. 2) including a processor and a control program.

The computer is operable on the electrical signal to create a hemostasisprofile corresponding to the measured clotting process. Additionally,the computer may include a visual display or be coupled to a printer toprovide a visual representation of the hemostasis profile. Such aconfiguration of the computer is well within the skills of one havingordinary skill in the art.

As will also be described, based upon an assessment of the hemostasisprofile, the computer, through its control program, may be adapted toprovide dosing recommendations. As shown in FIG. 3, the resultinghemostasis profile 20 is a measure of the time it takes for the firstfibrin strand to be formed, the kinetics of clot formation, the strengthof the clot (measured in millimeters (mm) and converted to shearelasticity units of dyn/cm²) and dissolution of clot. Table I, below,provides definitions for several of these measured parameters.

TABLE I R R time is the period of time of latency from the time that theblood was placed in the TEG ® analyzer until the initial fibrinformation. α a measures the rapidity of fibrin build-up andcross-linking (clot strengthening) MA MA, or Maximum Amplitude in mm, isa direct function of the maximum dynamic properties of fibrin andplatelet bonding via GPIIb/IIIa and represents the ultimate strength ofthe fibrin clot. LY30 LY30 measures the rate of amplitude reduction 30minutes after MA and represents clot retraction, or lysis.

Clinically, these measurements provide a vehicle for monitoringanticoagulation therapy (e.g. bivalirudin, heparin or warfarin, whichelongate the R parameter and reduce α.), thrombolytic therapy (e.g. tPA,streptokinase, urokinase, which increase LY30), effect ofantifibrinolytics (e.g. ε-amino-caproic acid (Amicar®), trasylol(aprotinin), tranexamic acid (TX), which reduce LY30), effect ofanti-platelet agents (e.g. abciximab (ReoPro®), eptifibatide(Integrilin®), tirofiban (Aggrastat®), which reduce MA), blood componenttransfusion therapy (which enhances the blood coagulation profile),thrombotic risk assessment in cancer and infection, high risk surgeryand other conditions which could possibly lead to excessive clotting(hypercoagulable conditions) or excessive bleeding (hypocoagulableconditions). In accordance with the invention then, the hemostasisanalyzer 10 is useful in testing the clinical efficacy of drug therapyto stop fibrinolysis, or the efficacy of thrombolytic drugs to monitorthrombolysis, efficacy of anti-platelet agents to monitor plateletinhibition, ischemic or bleeding complications.

Quantitatively, the hemostasis analyzer 10 and associated computer plotthe strength of the clot against time, where the onset of clotformation, the reaction time (R), is noted (FIG. 3). This plot alsoindicates the maximum clot strength (or rigidity), MA, of a bloodsample. MA is an overall estimate of platelet-fibrin GPIIb/IIIa bonding,which is used, for example, to guide post-operative blood platelet orfibrinogen replacement therapy.

The hemostasis parameters R, α, MA, and LY30 facilitate diagnosis ofvirtually any hemostasis disorder or evaluation of virtually anyhemostasis therapy. For example, as between platelets and fibrin alone,an abnormally low MA implies that there is an abnormality in bloodplatelets (i.e., a quantitative or functional defect) and/or anabnormality in fibrinogen content in the blood. However, by keepingfibrinogen level and platelet number constant, any change in MA wouldreflect changes in platelet function. Therefore, by testing the sameblood sample two ways, one with an anti-platelet agent and one without,the difference between the two MAs reflects the effect of theanti-platelet agent on platelet function. Similarly, isolating plateletcontribution allows a determination of fibrinogen content, i.e.,available functional fibrinogen.

Platelets play a critical role in mediating ischemic complicationsresulting in stroke and myocardial infarction. Inhibition of plateletfunction by anti-platelet agents (platelet-blocker drugs) such asaspirin, the antibody fragment c7E3 Fab, abciximab (ReoPro®), orclopidogrel, (Plavix®), can result in a dramatic reduction in the riskof death, myocardial infarction, or re-occlusion after percutaneouscoronary intervention (PCI) or intra-arterial thrombolytic therapy(IATT). Administration of excessive amounts of anti-platelet agentscould lead to life-threatening bleeding. Therefore, a precise estimateof platelet function inhibition in a given patient is very important forthe monitoring of the drug therapy because of the narrowrisk/therapeutic ratio with this class of drugs.

Using the above strategy, which keeps fibrinogen level and plateletnumber constant, it is possible to properly administer and monitoranti-platelet agents or modify their dosages, or to measure thecontribution of fibrin to MA (MA_(FIB)) and by subtraction to measurethe pure contribution of platelets to MA (MA_(p)) as MA_(p)=MA−MA_(FIB).

Therefore, in accordance with one possible preferred embodiment of theinvention, to properly monitor anti-platelet agents, the followingprocedure is followed:

-   -   1. The hemostasis analyzer 10, as it is commonly used, measures        platelet function (MA or MA_(P)) that is stimulated by thrombin,        a potent platelet activator that, as presently understood        activates the GPIIb/IIIa receptor site through proteolytically        activated receptors (PARs). To sensitize MA or MA_(P) to a small        inhibition of platelet function, platelet function is activated        by a platelet agonist such as ADP that indirectly activates the        GPIIb/IIIa receptor site (e.g., FIG. 11). Therefore, when        running blood samples in the hemostasis analyzer 10 in this        instance, formation of thrombin is inhibited with, for example,        sodium citrate, heparin, hirudin, bivalirudin, etc., and ADP is        used instead to activate the platelet function.    -   2. Unfortunately, thrombin is also involved in activating        fibrinogen to fibrin conversion. Having inhibited thrombin        formation in step 1, it is necessary to use another enzyme to        activate fibrinogen. Batroxobin (such as available under the        trade name Reptilase), whose sole function is to activate the        fibrinogen to fibrin conversion, is a suitable enzyme. The clot        is now stimulated by batroxobin (fibrinogen activation) and ADP        (platelet activation). The strength of the clot is measured by        MA, and the contribution of platelet function to the strength of        the clot is measured by MA_(P), as described above.    -   3. The clot that is formed by a fibrinogen activator like        reptilase and a platelet activator like ADP is typically weaker        than one developed by thrombin. Therefore, the torsion wire 15        described above may be selected to be sensitive to a weaker clot        and to be able to measure the changes in MA and MA_(P) due to        the small effect of anti-platelet agents such as ReoPro®.        Alternatively, activated Factor XIII (Factor XIIIa) may be        added. Factor XIIIa is believed to cause bonding modification of        crosslinked fibrin strands from hydrogen bonding to stronger        covalent bonding, enhancing clot strength.

Based on the above, the following protocol may be implemented:

-   -   1. Torsion wire modification of the hemostasis analyzer 10 as        necessary: by producing different strength torsion wires for        various sensitivities to shear force to adequately measuring the        effects of anti-platelet agents of various potencies may be        measured. The sensitivity of the torsion wire is generally        related to its gauge. For increased sensitivity, torsion wires        having gauges to sense clot sensitivity in a range from about        150 to about 1000 dyn/cm2 are suitable for adaptation to the        hemostasis analyzer described in the aforementioned U.S. Pat.        No. 6,225,236.    -   2. Batroxobin-triggered agonist-activated blood sample:        batroxobin (reptilase, Pentapharm) would be used (15 μl of        reconstituted batroxobin reagent) and pre-added to the cup 12 to        activate fibrinogen to fibrin. In addition to the batroxobin,        ADP (2 μM final concentration) would be pre-added to the cup 12        along with 10 μl of Factor XIIIa. 340 μl of thrombin inhibited        (e.g., citrated, heparinized, etc.) whole blood would be added        to the pre-warmed cup 12 containing batroxobin, ADP and Factor        XIIIa, and maximal clot strength would be measured providing an        assessment of fibrin contribution and ADP activated platelet        contribution to clot strength. In addition, clot strength of a        control sample, with complete inhibition of the platelet        contribution to clot strength (MA_(FIB)), would also be measured        with batroxobin and Factor XIIIa but no platelet activator being        added to the cup 12, providing an assessment of the contribution        of fibrin in the absence of the augmenting effect of platelets        to clot strength.

MA_(PB) is measured before the patient is treated with the anti-plateletagent and MA_(PA) is measured after treatment. Platelet inhibition dueto the drug effect will be computed as follows:MA _(PB) =MA _(B) −MA _(FIB)MA _(PA) =MA _(A) −MA _(FIB)Drug inhibition=MA _(PB) −MA _(PA)

For patients with high platelet activity (platelet hypercoagulability),who are at high risk of ischemic events, this method enables a means toadminister and monitor anti-platelet agents or modify dosage to attainan individualized therapeutic level of platelet inhibition and minimizethe patient's risk of ischemic events.

It will be appreciated by those having skill in the art that measuringclot strength as described above may require a torsion wire that issensitive to the typically weaker clot formed under conditions ofthrombin inhibition. However, different testing protocols may look toclots having strengths in ranges equal to or greater than typicalthrombin supported clots. In such cases the torsion wire 15 will beselected to be sensitive to such stronger clots. Torsion wires ofseveral gauges providing a range of sensitivities from about 100 dyn/cm²to 25,000 dyn/cm² therefore may be utilized.

It should be further appreciated that the invention has application tomeasuring other parameters of clot formation. For example, thehemostasis analyzer 10 measures the blood clotting process from the timeof test initiation until the initial fibrin formation, through clot ratestrengthening, and clot lysis. Therefore, in accordance with theinvention, it is possible to measure the effect of the presence ofheparin by evaluating the R parameter, which as described aboveindicates the inhibition in initial fibrin formation. It is alsopossible to measure the efficacy of drug therapy on thrombolyticactivity by observing the parameter LY30, which indicates the rate ofclot lysis.

Thrombin, which is the most potent platelet agonist, initiates theprocess of platelet aggregation. Thrombin is believed to act through aprotease activated receptor (PAR) receptor-mediated response, whichcauses the expression of GPIIb/IIIa receptors. The following is adiscussion of several factors and considerations related to thisprocess.

It is well-documented that there is considerable person-to-personvariability in the number of GPIIb/IIIa receptors per platelet and itsligand binding function. Furthermore, variable inhibition of GPIIb/IIIafunction, in part due to the differences in platelet count, may occurafter administration of a fixed, weight-adjusted dose of a plateletblocker. Higher risk patient subgroups, such as diabetic patientsundergoing PCI, may require greater doses of platelet inhibition than iscurrently being attained after weight-adjusted platelet blocker therapy,which at this time is not individualized to assure the attainment ofadequate GPIIb/IIIa receptor blockade. The potential for hemorrhagic orischemic events suggests the need for individualized assessment andprojecting of needed dosing to assure the attainment of a therapeuticlevel of receptor blockade, in real time. The apparatus and method inaccordance with the preferred embodiments of the invention provides thiscapability.

In contrast to direct platelet GPIIb/IIIa receptor inhibition agents,Plavix® (clopidogrel) is a platelet adenosine diphosphate (ADP) receptorantagonist, inhibiting a class of ADP receptors mediating activation ofplatelet GPIIb/IIIa receptors. Plavix® is taken orally, usually in theform of a loading dose of four 75 mg tablets followed by long-termtherapy of one 75 mg tablet per day prior to and after PCI or forpatients at high risk for ischemic events due to high platelet activity.The Plavix® algorithm dictates the same dosing regardless of patientweight or hemostatic profile. Consequently, treatment with Plavix® canresult in increased bleeding or lack of attainment of an adequatetherapeutic level of platelet inhibition. Therefore, there is a need toprescribe and monitor individualized dosing of both platelet GPIIb/IIIaand ADP receptor inhibition (PI) agents.

Thromboxane A₂ (TxA₂) activates the Thromboxane A₂ receptor. OnceThromboxane A₂ receptors are activated, they mediate the activation ofGPIIb/IIIa receptors. Cyclo-oxygenase is the enzyme necessary in theproduction of Thromboxane A₂, and is inhibited by non-steroidalanti-inflammatory drugs (NSAID).

The result of the activated coagulation protein is the fibrin strandwhich, together with activated platelets at GPIIb/IIIa, formsfibrin-platelet bonding (FIG. 1 b) to produce the final clot. Thereforefor fibrin-platelet bonding to occur or to take place, plateletGPIIb/IIIa receptors have to be activated. Therefore platelet agonistsare constructed toward activation of the GPIIb/IIIa receptor through PARreceptors, as by thrombin, or indirectly as by ADP and Thromboxane A₂.Consequently platelet inhibitor drugs are specifically targeted towardsinhibiting these agonists as illustrate in FIGS. 4-10.

With reference to FIGS. 4-10, a platelet 30 has a GPIIb/IIIa receptorsite 32, an ADP receptor site 34 and a TxA₂ receptor site 36. As shownin FIG. 4, addition of ADP agonist activates the ADP receptor site 34(illustrated by arrow 38), which activates the GPIIb/IIIa receptor site(illustrated by arrow 40). A platelet adenosine diphosphate (ADP)receptor antagonist, such as Plavix®, inhibits the ADP receptor site 34(arrow 42 in FIG. 5), and thus the GPIIb/IIIa site is not activated inresponse to the presence of the ADP agonist (phantom arrow 44).Therefore, in the presence of an ADP receptor antagonist, an ADP agonistonly activates platelets that are not inhibited. The result is areduction in clot strength, illustrated as the tracing MA_(pi) in FIG.11 as compared to the clot strength with complete platelet activationillustrated as the tracing MA_(kh) in FIG. 11.

ReoPro®, Integrilin®, and Aggrastat® inhibit the GPIIb/IIIa receptor 32directly. When the ADP agonist is added, it activates the ADP receptor34 (arrow 46 in FIG. 6) but is stopped at the GPIIb/IIIa receptor(phantom arrow 48). Therefore, in the presence of GPIIb/IIIa inhibitors,only the non-inhibited platelets will be activated by the ADP agonistresulting in correspondingly reduced clot strength, e.g., MA_(pi)illustrated in FIG. 11.

Thromboxane A₂ activates the platelet TxA₂ receptor 36 (arrow 50 in FIG.7), which in turn activates the GPIIb/IIIa receptor 32 (arrow 52).Arachidonic acid (AA) is a precursor to Thromboxane A₂ and is convertedto Thromboxane A₂ in the presence of Cyclo-oxygenase. Non-steroidalanti-inflammatory drugs (NSAID) inhibits Cyclo-oxygenase (arrow 54 inFIG. 8), and thus the GPIIb/IIIa site is also not activated in thepresence of a TxA₂ agonist (phantom arrow 56) resulting in acorresponding reduction in clot strength, e.g., MA_(pi) in FIG. 11.Therefore an AA platelet agonist can only activate the GPIIb/IIIa site32 when NSAID is not taken.

Thrombin is the enzyme that cleaves soluble fibrinogen into fibrinstrands. It is also the most potent platelet activator, stronglyincreasing the expression and activation of platelet GPIIb/IIIareceptors (arrow 58 in FIG. 9) through PARs receptors. It is believedthat PAR-1 and PAR-4 at least provide this expression, but others maycontribute. ReoPro®, Integrilin®, and Aggrastat® agents inhibitGPIIb/IIIa receptors responsive to ADP or TxA₂ agonists in vivo;however, they are not affective if administered in vitro as thrombinwill still result in GPIIb/IIIa activation (arrow 58 in FIG. 10).Certain hemostasis assays, such as the above-referenced TEG® assay, maybe arranged to produce thrombin in the process of clot formation. Theincreased expression and strong PARs receptor initiated activation ofplatelet GPIIb/IIIa by thrombin overcomes the GPIIb/IIIa inhibitors toprovide a full platelet activation clot strength (MA_(kh) in FIG. 11).In vivo, thrombin escaping into the circulation is immediately inhibitedby endogenous anti-thrombin agents or by forming a complex molecule withendothelial thrombomodulin, localizing the hemostatic response to thesite of injury. Ex vivo, for example when blood is placed in a testingapparatus, thrombin generation is continuous throughout the clotformation process. Due to the absence of endothelium, inhibitoryactivity is limited to the endogenous anti-thrombin agents in the sampleand remains viable to cleave fibrinogen and mediate activation ofGPIIb/IIIa receptors. Thus, as described herein, thrombin is suppressedto allow isolation and evaluation of other hemostasis factors andtherapies.

With the foregoing discussion, it is therefore possible to furtherspecify a protocol for monitoring platelet inhibitors, such asGPIIb/IIIa, ADP and Thromboxane A₂ platelet inhibitors. Referring toFIG. 12, an apparatus 10′ may include a plurality of hemostasis analysisdevices, such as the hemostasis analyzer 10 shown in FIG. 2, and fourare shown as devices 10 a, 10 b, 10 c and 10 d, to respectively testcorresponding blood samples 13 a, 13 b, 13 c and 13 d.

A first sample 13 a of heparinized whole blood is prepared and loadedinto the first hemostasis analyzer 10 a. A fibrin activator 17, such asa combination of batroxobin and Factor XIIIa, is added to the sample cup12. The activator 17 may be pre-added to the sample cup 12 or the cup 12may be treated with the activator 17. Alternatively, the activator 17may be added to the blood sample 13 a after it is added to the cup 12.About 10 μl of activator 17 is added to a 340 μl blood sample. The assayis completed and the resulting clot strength is measured. This clotstrength may be referred to as MA_(f), as it represents only thecontribution of fibrin with substantially no platelet activation in viewof the absence of any platelet agonist.

A second sample 13 b of heparinized whole blood is prepared and isloaded into the second hemostasis analyzer 10 b. It should be noted thata single cell hemostasis analyzer may be used serially for each of theassays, more than one single cell analyzer may be used, or multi-cellanalyzers may be used. For example, the TEG® hemostasis analyzer instandard configuration has two testing cells, which may operatesimultaneously. Two TEG® hemostasis analyzers may be used to performeach of the four assays according to this exemplary protocol or one TEG®hemostasis analyzer may be used. Moreover, the two TEG® hemostasisanalyzers may be networked, making in essence a single four cell testingdevice.

For the second sample 13 b, the activator 17 is added to the sample, andan ADP agonist 18 is also added in appropriate proportion to the secondsample 13 b. For example, 2 μM of ADP agonist may be added to a 340 μlsecond sample of heparinized whole blood. The assay is completed and theresulting clot strength is measured. This clot strength may be referredto as MA_(pi1), as it represents the contributions of fibrin andplatelets uninhibited by any administered ADP platelet inhibitionagents.

A third sample 13 c of heparinized whole blood is prepared and is loadedinto a third hemostasis analyzer 10 c. The fibrin activator 17 added tosample 13 c, and a Thromboxane A₂ agonist 19 is added in appropriateproportion to the third sample 13 c. For example, 10 μl of ArachidonicAcid (AA) may be added to a 340 μl third sample of heparinized wholeblood. The assay is completed and the resulting clot strength ismeasured. This clot strength may be referred to as MA_(pi2), as itrepresents the contributions of fibrin and platelets uninhibited byadministered TxA₂ platelet inhibition agents.

A fourth sample 13 d of heparinized whole blood is prepared and isloaded into a fourth hemostasis analyzer 10 d. For the fourth sample 13d, heparinase 21 and kaolin 22 are used to neutralize the effect of theheparin in the fourth sample 13 d and accelerate the thrombinexpression, respectively. The assay is completed and the resulting clotstrength is measured. This clot strength may be referred to as MA_(kh),and it measures the maximum MA with platelet activation due to the useof heparinase and kaolin to neutralize the heparin in the sample 13 dand to enable the production of thrombin which activates via the PARsreceptors the GPIIb/IIIa receptors regardless of the inhibition of TxA₂,ADP, and GPIIb/IIIa receptors.

The value MA_(pi)−MA_(f) measures the unique contributions of theuninhibited platelets by PI agents where platelet inhibition can be bythe in vivo administration of agents, such as, Reopro®, Aggrastat®,Integrilin®, Plavix® and NSAIDs. A percentage reduction in MA due toplatelet inhibition is then calculated for each of MA_(pi1), andMA_(pi2) according the equation:Percent Platelet Activation=[(MA _(pij) −MA _(f))/MA _(kh) −MA_(f))]*100,where the value MA_(kh) −MA _(f) measures the unique contributions ofthe fully activated platelets. Thus, the medical practitioner mayobserve the effect of platelet inhibition therapy, isolated intocomponent effects, and make dosing recommendations accordingly.Alternatively, percent platelet inhibition may be determined as100-[(MA_(pij) −MA _(f))/MA_(kh) −MA _(f))]*100.

It will be appreciated that the sample cups 12 in the foregoing assaysmay be prepared in advance to contain the appropriate agents inaccordance with the foregoing described protocol. In that regard, thesample cups may be color coded to identify the particular agentscontained within the cup. Sets of sample cups 12 may be packaged tofacilitate the assays. Still further, the agent materials, e.g., theactivator 17, may be distributed in color coded vials for ease ofidentification and for loading into the sample cups 12 either before orafter the corresponding blood sample is loaded into the sample cup.

Assays in accordance with various preferred embodiments of theinvention, such as the examples described above, may employ thrombinsuppression, via in vitro or otherwise introduction of a suitablethrombin inhibitor, such as heparin. However, with thrombin suppressionand a corresponding absence of thrombin contribution to hemostasis,e.g., cleavage of fibrinogen to form fibrin, additional reagents arerequired to compensate for the lack of thrombin and its contributingfactors beyond platelet activation.

As noted, in accordance with preferred embodiments of the invention, itis possible to evaluate patient specific dosing and efficacy ofvirtually any hemostasis therapy by measuring and considering one ormore of the hemostasis parameters. For example, it may be possible tomonitor an anticoagulation therapy such as bivalirudin, heparin,warfarin or the like.

One exemplary protocol utilizes a sample, e.g., sample 13, that isobtained from a subject prior to administration of the anticoagulationtherapy. Another sample is obtained at a first time period followingadministration of an anticoagulation therapy such as a direct thrombininhibitor therapy like bivalirudin, heparin or warfarin. Each sample maybe obtained into sodium citrate.

The sodium-citrated samples are recalcified, and an additional reagentis provided such as a prothrombin activator such as ecarin. Thehemostasis analyzer 10 is used to measure the time to initial clotformation, R. FIG. 13 illustrates data for a sample of subjects showingtime to initial clot formation R before in vivo administration of theanticoagulation therapy and after in vivo administration of theanticoagulation therapy. FIGS. 14 a and 14 b illustrate tracings and,respectively, showing a typical set of hemostasis parameter data beforein vivo administration of the anticoagulation therapy and after in vivoadministration of the anticoagulation therapy. As the tracings andillustrate, apart from the change in the time to initial clot formationR, the tracings are otherwise typical, i.e., the rate of clot formationα, the clot strength MA and lysis, LY30 are otherwise within expectednorms. Importantly, there exists correlation between the delay ininitial clot formation and the dosing of the anticoagulation therapy.For the example illustrated in FIGS. 13, 14 a and 14 b , the delay inonset of clot formation shows a direct relationship to bivalirudindosing. As described above, therefore, the whole blood hemostasisanalyzer, such as the TEG® hemostasis analyzer, may be used to evaluatepatient specific dosing of particular anticoagulation therapies forpatients with enzymatic hypercoagulability.

For patients with accelerated initial clot formation (R, indicatingenzymatic hypercoagulability as a result of rapid thrombin generation)who are at high risk of ischemic events, the method enablesadministration and monitoring of anticoagulation agents or modifcationof dosage to attain an individualized therapeutic level ofanticoagulation and minimize the patient's risk of ischemic events.

Hemostasis is a very complex process, with multiple interactions amongfactors which include the procoagulant and anticoagulant proteins andcellular elements. Nothing in the hemostasis system is static or inisolation. Therefore, to reduce the probability of ischemic events, onehas to look at the source of the prothrombotic state and determinewhether it is due to enzymatic or platelet hypercoagulability or both.On an individual basis, one has to anticoagulate the patient, administeranti-platelet agents, or both in order to reach a balanced hemostasiswhere both ischemic events and bleeding are minimized.

The foregoing embodiments are described as generally applicable for postPCI patients, to screen for and prevent recurrence of ischemic events.However, the same procedure may be employed to screen initially forischemic risk. Thus, the methodology may be employed in a prophylacticmanner to assess initial risk of ischemic event and to inform apreventive therapy, such as the administration and dosage ofanticoagulation agents.

The hemostasis analyzer 10 may include or be linked to a database. Thedatabase may contain correlation and/or normative data to which instantsample results may be compared by the processor to determine a patientspecific dosing of the hemostasis therapy. The processor may beconfigured to make such a determination or, for example, when thedatabase is remotely located from the hemostasis analyzer 10, aprocessor located with the database may be configured for such purpose.

It is presently understood that human platelets are activated bythrombin cleavage of PAR-1 and/or PAR-4 causing platelet aggregation(platelet-fibrin(ogen) bonding via GPIIb/IIIa receptors) and secretion.Antibody blockade of both PAR-1 and PAR-4 prevents virtually allthrombin-mediated platelet aggregation and secretion. This suggests thatPAR-1 and PAR-4 mediate most, if not all, of the thrombin mediatedsignaling in human platelets. With PAR-1/PAR-4 inactivation and thethrombin's active site still intact, thrombin's pro-coagulant actions,e.g., cleavage of fibrinogen to form fibrin, activation of the serineprotease FXI (Factor XI) and transglutaminase FXIII and othernon-enzymatic coagulation factors remain intact. Thus, in the foregoingprotocols, suppression of thrombin's platelet activating function byantibody blockade of PAR-1 and PAR-4 may be employed in theanti-platelet assay. At the same time, additional reagents are notrequired to preserve the pro-coagulant action of thrombin, e.g., cleavefibrinogen to fibrin by batroxobin and activate Factor XIII to FactorXIIIa for fibrin cross-linking and the like in the anti-platelet assay.

The invention has been described in terms of several preferredembodiments. One of skill in the art will appreciate that the inventionmay be otherwise embodied without departing from its fair scope, whichis set forth in the subjoined claims.

1. A method of preparing an individualized ischemic event riskassessment and individualized treatment for a subject, the methodcomprising: evaluating a first blood sample portion obtained from asubject to determine a first clot characteristic related quantitativeindication of hemostasis of the first blood sample portion; determininga parameter indicative of the risk of the subject having an ischemicevent based upon the first clot characteristic related quantitativeindication of hemostasis and determining a treatment for the subjectbased upon the parameter; evaluating a second blood sample portionobtained from the subject post in vivo administration of a coagulationtherapy to determine a second clot characteristic related quantitativeindication of hemostasis of the second blood sample portion; anddetermining a post therapy parameter indicative of the risk of thesubject having an ischemic event based upon the second clotcharacteristic related quantitative indication of hemostasis anddetermining an efficacy of the coagulation therapy for the subject basedupon the parameters; wherein evaluating each of the first sample portionand the second sample portion comprises emulating venus flow for each ofthe first blood sample portion and the second blood sample portion. 2.The method of claim 1, wherein each of the first blood sample portionand the second blood sample portion are prepared including in vitroadministration of an activator.
 3. The method of claim 1, wherein thefirst and second quantitative indications comprise one of initial clotformation, clot strength, clot elasticity, rate of clot formation orrate of clot lysis.
 4. The method of claim 1, comprising sequentiallytesting the first blood sample portion and the second blood sampleportion.
 5. The method of claim 1, comprising providing a first andsecond hemostasis analyzer, and sequentially testing the first and thesecond blood sample portions.
 6. The method of claim 5, wherein thefirst and second hemostasis analyzers comprise first and second testingcells of a single hemostasis analyzer.
 7. The method of claim 1,comprising comparing the first and the second clot characteristicquantitative indications to correlation data and determining a dosingparameter of the coagulation therapy in view of the correlation data. 8.An apparatus for preparing an individualized ischemic event riskassessment and providing individualized hemostasis therapy for a subjectcomprising: a hemostasis testing cell for testing a first blood sampleportion obtained from a subject pre-treatment to determine a first bloodsample clot characteristic related haemostasis parameter for the firstportion; means for providing a first parameter indicative of the risk ofthe subject having an ischemic event based upon the first blood sampleclot characteristic related hemostasis parameter; means for providing anindication of an individualized treatment for the subject based upon thefirst parameter; the hemostasis testing cell further for testing asecond blood sample portion obtained from the subject - post in vivoadministration of a coagulation therapy to determine a second bloodsample clot characteristic related haemostasis parameter for the secondportion, so that during testing of the second portion the second bloodsample clot characteristic related hemostasis parameter isdistinguishable from other blood sample hemostasis characteristics; andmeans for providing a second parameter indicative of the risk of thesubject having an ischemic event based upon the second blood sample clotcharacteristic related hemostasis parameter and the second parameterbeing indicative of the efficacy of the coagulation therapy; whereintesting of each of the first sample portion and the second sampleportion comprises emulating venus flow for each of the first bloodsample portion and the second blood sample portion.
 9. The apparatus ofclaim 8, wherein the testing cell comprises first and second testingcells of a single testing apparatus.
 10. The apparatus of claim 8further comprising a processor for evaluating the first and second bloodsample clot characteristic parameters.
 11. The apparatus of claim 10,wherein the processor is linked to a database comprising correlationdata, the processor being configured to determine a dosing parameter ofthe coagulation therapy in view of the correlation data.
 12. Theapparatus of claim 11, wherein the database is remote from theapparatus, the apparatus being linked to the database via acommunication link or network.